The present invention relates generally to tissue arrays used in histology for medical and biological research as well as in medical diagnosis and treatment of disease.
The virtual explosion of knowledge in biotechnology in recent years has caused an enormous demand for tissue microarray blocks. These microarray blocks contain tissue samples that provide multiple on-slide tissues that are useful for a variety of purposes.
The prior art techniques for producing tissue microarray blocks typically involve transferring of solid core samples from a donor tissue sample into a blank recipient wax block. The thickness of these prior art solid core samples is determined by the thickness of the donor tissue. As an example, if the donor tissue has a thickness of 2 mm, the solid core sample transferred into a well of a prior art tissue microarray block will have that same 2 mm thickness. When sliced into sections for microscopy, that particular tissue sample can produce about 200 slides. In a prior art tissue microarray block having a plurality of wells containing different solid tissue samples with differing thicknesses, each well will produce a different number of “daughter” slides. This results in an inefficient technique, since the well with the thinnest solid core sample will determine the ultimate number of slides obtainable from the block. Therefore, in the prior art technique, the number of obtainable slides is limited, and there is also a waste of donor tissue.
There is clearly a dire need for a more efficient technique of producing tissue microarray blocks wherein each well of the block contains a solid tissue sample having a depth not determined by the thickness of the solid donor tissue. In fact, for the purposes of immunohistologic diagnosis, the College of American Pathologists has recommended multiple on-slide tissue controls for every patient (Commission On Laboratory Accreditation, Laboratory Accreditation Program Anatomic Pathology Checklist, S. Sarewitz, MD, Editor, September 2007, pp. 45-46). Given the enormous demand for tissue microarrays (TMAs) that this would represent, this goal has not yet been realized, and a more efficient method for TMA production is required. The present invention provides for more efficient TMA production and, in addition, achieves a “high-yield” tissue microarray block capable of producing many times more slides from a single block than the prior art!
The manner in which the present invention achieves these dramatic results is elegant in its simplicity. Rather than using solid, rod-shaped coring samples, one embodiment of the present invention “minces” the solid donor tissue into fragments. The fragments are embedded in a liquefied carrier having a low melting point, such as wax. The solid, minced fragments are evenly distributed throughout the length of the molten, liquefied carrier, forming a two-phase (i.e. solid and liquid) mixture. The two-phase mixture may be formed into the shape of a “deep well” and transferred into a microarray block while the carrier remains molten. In another embodiment, the two-phase mixture is cooled to solidify the carrier prior to being transferred into a “deep well,” such as 17 mm long and 2 mm diameter, for example. By using this “mincing” technique together with forming the desired shape of a deep well, both objectives described above are achieved along with reduced waste of donor tissue. First, the thickness of the solid donor tissue transferred to each well is not limited to the thickness of the original donor tissue. Second, and equally important, the thickness of the tissue samples transferred into the wells of the block is uniform and of much greater thickness than the prior art. The result of the present invention is an efficient, cost effective, high quality and high yield tissue microarray block capable of producing 1,000 to 2,000 replicate slides, 5 to 10 times more than the prior art!
Alternatively, rather than “mincing” solid donor tissue, homogenous tissue derived from cell culture may be used and, after appropriate fixation, suspended in a liquid carrier and transferred in liquid form into a deep well. In a distinct version of the technique, the mixture of cell culture material and carrier can be allowed to solidify within a duct of cross-sectional size and shape approximately equal to that of the deep well, and subsequently transferred in solid form into the deep well by simple extrusion from said duct, via the simple application of force by a plunger or other means. Both of these techniques differ from prior art techniques using cell cultures to form TMAs, in that the prior art solidifies the mixture of cell culture and carrier (by centrifugation, re-suspension in a carrier and solidification of the mixture) and then takes core samples of the resulting solid material (Moskaluk and Stoler, “Agarose Mold Embedding of Cultured Cells for Tissue Microarrays,” Diagnostic Molecular Pathology 11(4): 234-238, 2002; also Montgomery et al, “A Novel Method for Making ‘Tissue’ Microarrays From Small Numbers of Suspension Cells,” Applied Immunohistochemistry & Molecular Morphology, 13(1): 80-84, 2005).
Another aspect of the present invention is a novel system for forming the two-phase mixture of minced solid donor tissue into elongated columns (17 mm long and 2 mm in diameter, for example) of a meltable carrier such as wax. An elongated passageway is formed in a body of thermally conductive, preferably transparent material. A source of sufficient heat is applied to the passageway in order to maintain part or all the length of the passageway above the melting-point temperature of the carrier substance. Paraffin wax is a good example of a suitable carrier, but other substances can also be used. Molten carrier is transferred into the elongated passageway by one or more of several means: (a) the pure carrier substance may be introduced in the molten state from a heated dispenser; (b) the pure carrier substance may be introduced in the solid state, relying on the temperature of the passageway to melt the carrier; (c) tissue samples may be embedded in the carrier substance, which melts due to the temperature of the passageway. Option (c) is often preferable in the case of minced solid tissue fragments. In any case, the minced solid donor tissue fragments, or other tissue constituents, such as obtained from cell cultures, are introduced to the passageway along with the molten carrier substance, thus creating a two-phase mixture (suspension of biological material in liquid carrier). At the entrance of the passageway may exist a collecting receptacle or “hopper,” also heated, to facilitate the melting of carrier and the mixing of biological material in liquid carrier. A duct originates in the hopper and intersects the passageway. With or without the use of a hopper, the liquid carrier and biological material are inserted into the passageway. Some means of propelling this two-phase mixture through the passageway is implemented, such as: (a) pressing with a plunger that contacts the mixture and propels it similarly to the action of a syringe; (b) the application of hydraulic pressure to the passageway after sealing any side-ports such as the intersecting duct from a hopper; (c) gravity-driven flow of the mixture; (d) propulsion of the mixture by direct contact with an auger feeder within the passageway. It is the liquid state of the carrier during this step that enables such a range of possible propulsion mechanisms, and also in general enables the mixture to flow through a duct with curvature, converging and/or diverging sections. During its travel through the passageway, the velocity and temperature of the mixture are controlled by the speed or pressure of the propulsion mechanism and, if necessary, the management of heat applied to the passageway, order to form the mixture into a dense column and to control the state of liquidity of the carrier substance. Some degree of cooling, in order to increase the viscosity of the liquid carrier, or even to solidify it, can be advantageous near the end of the passageway. The compressed tissue column is then extruded out of the elongated passageway into a well within a recipient block.
In the case where the biological material for the microarray comes from a solid human or animal tissue source, the mincing step must be done in order to obtain a sufficiently pure tissue of interest, and with techniques appropriate to the type of tissue and to the required orientation of tissue structures in the product slides and hence in the microarray block. In summary, the process of mincing depends on the type of tissue. In many cases, skilled dissection by hand is required, but in other cases mincing can be done by an automated fragmenting process such as with sharp rotating blades. As used herein and in the claims, the term “mincing” is used in a broad sense to include (a) simple fragmentation of a solid tissue sample that has been previously selected for its purity of tissue type, when purity of tissue type is sufficient for the purposes of the resulting microarray analysis; (b) careful dissection into fragments of a more precisely specified biological and physical structure, when such precision is necessary in order that the required biological and physical structures are present in the resulting microarray. Two illustrative examples are provided in the next paragraph.
Careful dissection may be required to harvest the tissue of interest at various steps depending on the tissue chosen. Illustration is provided by way of two examples that demonstrate some of the range of precision required for different tissue types. Colonic epithelium is harvested from a colonic resection specimen, preferably one that is non-fixed. The colon is opened longitudinally, using sharp dissection (scalpel blade) to separate the mucosa from the underlying fibromuscular soft tissue. Sheets of colonic mucosa are obtained in this manner and are cut into small sections of appropriate size for placing in plastic histology cassettes (up to about 20 mm×10 mm×4 mm). The sectioned mucosa is submitted in cassettes for routine histologic fixation and “processing” (dehydration and embedding in wax). After processing, the wax shroud is melted away, and the solid tissue is minced with a scalpel blade. Minced fragments should be on the order of 2 mm×2 mm×2 mm. The size of the fragments and their orientation can be important in some circumstances. For instance, skin fragments should have a long axis parallel to the plane of the dermal-epidermal junction, and this axis should be loaded parallel to the long axis of the “barrel” of the injector, such that the dermal-epidermal junction is ultimately visualized in cross-section on the microscope slide.
A further aspect of the present invention is a novel apparatus for forming the recipient (blank, open-well) microarray blocks. The apparatus provides an efficient system for forming the required array of much deeper wells than the wells of the prior art. The apparatus includes a means of easily varying the number and configuration of wells by removable/configurable pins in a mold device. This is useful for customizing the size and shape of the array. However, it should be noted that a casting technique is not the only way to produce the recipient microarray blocks. For example, an alternative and efficient method would be to use a drilling, reaming, and/or milling method for creating deep wells in an initially solid block of machinable wax, using a manual or computer numeric controlled (CNC) milling or drilling machine.
The prior art techniques of forming tissue arrays are summarized in the article by Eguiluz et al entitled “Multitissue array review: A chronological description of tissue array techniques, applications and procedures” published in Pathology-Research and Practice 202 (2006) at pp. 561-568 and in U.S. Pat. Nos. 6,103,518 and 7,029,615.
As noted above, the first embodiment of the present invention differs significantly from the prior art by using minced solid tissue fragments to form a two-phase mixture with a molten carrier such as wax. The prior art uses core samples of solid donor tissue. The mincing (and suspension) technique inherently allows the use of more uniform length wells and deeper wells than the prior art with less waste of donor tissue.
A primary object of the invention is to provide a high yield tissue microarray block wherein minced solid donor tissue fragments are suspended in a meltable or molten carrier such as wax.
An object of a second embodiment of the invention is to construct microarrays where the source of biological material comes from another source such as cell culture.
A further object of the invention is to provide a tissue microarray block wherein each well has an increased depth not determined or limited by the thickness of the donor tissue.
Another object of the invention is to provide a method and apparatus for forming elongated columns of a meltable carrier, such as wax, wherein suspended fragments of minced solid donor tissue are distributed evenly over the length of the column.
Another object is to provide an apparatus for forming a tissue microarray block having a plurality of wells wherein each well has a depth significantly greater than prior art wells.
Other objects and advantages will become apparent from the following description and drawings.